Analysis Of An Actin Binding Guanine Exchange Factor, Gef8, And Actin Depolymerizing Factor In Arabidopsis Thaliana.

ABSTRACT ANALYSIS OF AN ACTIN BINDING GUANINE EXCHANGE FACTOR, GEF8, AND ACTIN DEPOLYMERIZING FACTOR IN ARABIDOPSIS THALIANA. April, 2010 Aleksey Chudnovskiy, B.S., UNIVERSITY OF MASSACHUSETTS AMHERST M.S., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor Alice Y. Cheung Polarized cell growth is a fundamental biological process that is tightly regulated spatially and temporally. In plants the key systems to study polar cell growth are pollen tubes and root hairs. In recent years a lot of work has focused on elucidating the mechanisms that mediate this process. The actin cytoskeleton plays a key role in polarized cell growth. Different studies in plant and animal models show that signaling mediated through small GTP-binding proteins is a common theme in actin signaling. In recent years many groups have shown that small GTP binding proteins regulate actin dynamics through the activity of Actin Binding Proteins (ABP). In this study I explored the function of two ABPs from Arabidopsis Thaliana: Actin depolymerizing factor (ADF) and a novel actin-binding guanine exchange factor (GEF). I used Arabidopsis protoplasts as a system to study the function of these proteins. We showed through over-expression of the GFP labeled GEF8 under the constitutively active 35 S promoter, that GEF8 labels the prominent cable like structures inside the cell. Using actin and tubulin binding drugs such as Latrunculin and Oryzalin we showed that GEF8 labels actin cables. Using the Yeast 2 Hybrid system we determined that GEF8 binds actin filaments directly. We established that GEF8 interacts with actin through the unique N terminus of the protein. Finally, using the Basic Local Alignment Search tool we showed that the N terminus of GEF8 is homologous to the Actin Binding Protein 140, a well-established protein marker in Yeast. ADF is an established key regulator of the actin cytoskeleton. Much is known about ADF regulation in animal systems. In plants it has been shown that the small Rho type GTP binding proteins, called RAC/ROPS, regulate ADF activity and that overexpression of RAC/ROPs causes the inactivation of ADF through the phosphoryaltion on Serine 6. However, little is known about the proteins that transduce the signal from small GTP binding proteins to the ADF. Here we show some evidence that upon overexpression of Ric 4 (a RAC/ROP effector known to play a role in actin polymerization), ADF gets displaced from the filament. Moreover, ADF is known to be inactivated by phosphorylation at Ser6; the kinase responsible for this phosphorylation has not been identified in plant. We observed that over-expression of Calcium Dependent Protein Kinase 16 (CDPK16) in protoplasts also induced dissociation of ADF from actin cables. These results suggest that both of RIC4 and CDPK16 may play a role in the pathways that regulate ADF activity

Innate lymphoid cells integrate stromal and immune signals to enhance antibody production by splenic marginal zone B cells

Innate lymphoid cells (ILCs) regulate stromal, epithelial and immune cells, but their impact on B cells remains unclear. We identified RORγt + ILCs nearby the marginal zone (MZ), a splenic compartment containing innate-like B cells that respond to circulating T cell-independent (TI) antigens. Spenic ILCs established a bidirectional crosstalk with MAdCAM-1 + marginal reticular cells by providing tumor necrosis factor (TNF) and lymphotoxin, and activated MZ B cells via BAFF, CD40 ligand and the Notch ligand, Delta-like 1. Splenic ILCs further helped MZ B cells and their plasma cell progeny by co-opting neutrophils through the release of GM-CSF. Consequently, ILC depletion impaired both pre- and post-immune TI antibody responses. Thus, ILCs integrate stromal and myeloid signals to orchestrate innate-like antibody production at the interface between the immune and circulatory systems

Rapid monocyte kinetics in acute myocardial infarction are sustained by extramedullary monocytopoiesis

IL-1b signaling augments continued splenic monocyte supply during acute inflammation

Monocyte Subset Dynamics in Human Atherosclerosis Can Be Profiled with Magnetic Nano-Sensors

Monocytes are circulating macrophage and dendritic cell precursors that populate healthy and diseased tissue. In humans, monocytes consist of at least two subsets whose proportions in the blood fluctuate in response to coronary artery disease, sepsis, and viral infection. Animal studies have shown that specific shifts in the monocyte subset repertoire either exacerbate or attenuate disease, suggesting a role for monocyte subsets as biomarkers and therapeutic targets. Assays are therefore needed that can selectively and rapidly enumerate monocytes and their subsets. This study shows that two major human monocyte subsets express similar levels of the receptor for macrophage colony stimulating factor (MCSFR) but differ in their phagocytic capacity. We exploit these properties and custom-engineer magnetic nanoparticles for ex vivo sensing of monocytes and their subsets. We present a two-dimensional enumerative mathematical model that simultaneously reports number and proportion of monocyte subsets in a small volume of human blood. Using a recently described diagnostic magnetic resonance (DMR) chip with 1 µl sample size and high throughput capabilities, we then show that application of the model accurately quantifies subset fluctuations that occur in patients with atherosclerosis

Host-Protozoan Interactions Protect from Mucosal Infections through Activation of the Inflammasome

While conventional pathogenic protists have been extensively studied, there is an underappreciated constitutive protist microbiota that is an integral part of the vertebrate microbiome. The impact of these species on the host and their potential contributions to mucosal immune homeostasis remain poorly studied. Here, we show that the protozoan Tritrichomonas musculis activates the host epithelial inflammasome to induce IL-18 release. Epithelialderived IL-18 promotes dendritic cell-driven Th1 and Th17 immunity and confers dramatic protection from mucosal bacterial infections. Along with its role as a protistic'' antibiotic, colonization with T. musculis exacerbates the development of T-cell-driven colitis and sporadic colorectal tumors. Our findings demonstrate a novel mutualistic host-protozoan interaction that increases mucosal host defenses at the cost of an increased risk of inflammatory disease

Ex-vivo Nuclear Magnetic Resonance generates an enumerative mathematical model for monocyte subsets.

<p>A. Representative NIH-color coded map generated from T₂-weighted Magnetic Resonance Imaging. Data show equal number of CD16^lo, CD16^hi monocytes and other leukocytes labeled with two CLIO-MCSFR (left panel) and CLIO (right panel) concentrations. B. T₂ measurements detected with a conventional benchtop-relaxometer. Data show equal number of CD16^lo, CD16^hi monocytes and other leukocytes labeled with two CLIO-MCSFR (left panel) and CLIO (right panel) concentrations. N = 3–5. Mean±SEM. C. T₂ changes detected with a diagnostic magnetic resonance (DMR) chip. Data show increasing number of CD16^lo, CD16^hi monocytes and other leukocytes labeled with one CLIO-MCSFR (left panel) and CLIO (right panel) concentration. N = 3. Mean±SEM. D. Two-dimensional T₂ map derived from data in C to simultaneously enumerate total monocyte numbers and subset proportions. Model combines T₂ changes for CLIO-MCSFR (x-axis) and CLIO (y-axis). Changes in predicted monocyte number are demarcated with vertical lines while the rainbow region defines monocyte subset fluctuations.</p

Human monocyte subsets differ phenotypically and functionally.

<p>A. Flow cytometry dot plots show forward scatter (FSC) versus side scatter (SSC) of mononuclear cells obtained from fresh blood. A monocyte gate is drawn and monocyte subsets are identified according to their CD14 and CD16 expression profile. B. Histograms depict MCSFR expression of CD16^lo monocytes, CD16^hi monocytes and other cells (mostly lymphocytes). C. Table summarizes relative expression profiles of selected markers for CD16^lo, CD16^hi monocytes and other cells. D. Representative histograms and H&E cytospin preparations show CD68 expression and morphology of CD16^lo, CD16^hi monocytes and other cells freshly isolated (□) or after in vitro culture for 6 days with LPS/IFNγ. E. Bar graph depicts ex vivo phagocytosis of fluorescently labeled latex beads in CD16^lo, CD16^hi monocytes and other cells (n = 4).</p

Host-Protozoan Interactions Protect from Mucosal Infections through Activation of the Inflammasome

"While conventional pathogenic protists have been extensively studied, there is an underappreciated constitutive protist microbiota that is an integral part of the vertebrate microbiome. The impact of these species on the host and their potential contributions to mucosal immune homeostasis remain poorly studied. Here, we show that the protozoan Tritrichomonas musculis activates the host epithelial inflammasome to induce IL-18 release. Epithelialderived IL-18 promotes dendritic cell-driven Th1 and Th17 immunity and confers dramatic protection from mucosal bacterial infections. Along with its role as a "protistic'' antibiotic, colonization with T. musculis exacerbates the development of T-cell-driven colitis and sporadic colorectal tumors. Our findings demonstrate a novel mutualistic host-protozoan interaction that increases mucosal host defenses at the cost of an increased risk of inflammatory disease.

Magnetic nano-sensors enumerate monocyte subset variations that occur in atherosclerotic patients.

<p>A. Representative flow cytometry dot plots of monocyte subsets from healthy volunteers and patients with documented coronary artery disease (CAD). Numbers depict percentage of subsets in both groups. Mean±SEM. B. Plots depict percentage (left plot) and absolute numbers (right plot) of CD16^lo and CD16^hi monocytes from healthy volunteers (green dots) and patients with CAD (red dots). C. Validation of the enumerative mathematical model with varying number and percentage of monocyte subsets. Data show goodness of fit of defined numbers of monocytes alone (left panel), other leukocytes (middle-left panel) and combinations of leukocytes and monocytes (right two panels). Different proportions of monocyte subsets (CD16^lo/CD16^hi (%)) are color-coded and their fit is depicted on the two-dimensional T₂ maps.</p